First prime and then pull – the novel immunization approach

Some areas of our body enjoy a special status as far as the immune reaction is concerned. Anatomical entities like the gut or female genital tract as well as other mucosal surfaces do not support the same extend of protective response compared to many non-mucosal tissues. This exclusion is crucial to avoid the unwanted inflammation in places that are regularly exposed to the outer environment but sometimes it may present a problem when there is the need to elicit the strong protective response at such privileged site. I have found an interesting report which applies the novel vaccination strategy aimed to enhance the protection against herpes simplex virus 2 which being the virus transmitted through the contact with infected body fluids often enters the body through the genital organs. The innovation that this report introduces consists of double treatment (“prime and pull”) which bypasses the restrictive entry of memory T cells into the vaginal mucosa.

The link: http://www.nature.com/nature/journal/v491/n7424/full/nature11522.html

The mentioned “prime and pull” strategy is the subcutaneous immunization with an attenuated strain of HSV-2 (prime) which is followed by the topical application of chemokines CXCL9 and CXCL10 to the vaginal mucosa (pull). Authors follow the localization of CD8 T cells that recognize an epitope within one of HSV-2 glycoproteins and activated CD4 T cells to show that the distal immunization event plus the localized chemokine treatment provokes the significant recruitment of activated lymphocytes to the vagina whereas the immunization alone has much weaker effect. Interestingly, this recruitment is specific to CD4 and CD8 lymphocytes and does not encompass other cell types that express the relevant chemokine receptor CXCR3.

Is the “prime and pull” approach able to provide the longstanding and reliable protection? Data demonstrate that CD8 T cells (but not CD4 T cells) are retained at vaginal mucosa after the primary response period is over. Most importantly the “prime and pull” treatment may be indeed superior in enforcing the better protective immunity to HSV-2 challenge than the immunization alone. Investigators also ask about the mechanism by which the protection is delivered by the “prime and pull” strategy. It appears that this application can prevent the virus from entering the nervous system where HSV-2 propagates past the mucosal stage of infection.

What will be the future of “prime and pull”, though? The pros are obvious – there is the simple method to enhance the mucosal migration of protective lymphocytes without the “ugly face” of immunity which in this case would be the excessive inflammation at the sensitive anatomical location. Authors speculate about the future applications ranging from HIV protection to solid tumors treatment. The method itself may also be developed as in the discussed paper it provides the optimal protection only in conjunction with the adoptive transfer of virus-specific lymphocytes. The “pull” works as well with the endogenous population of CD8 T cells; however, the protection is suboptimal in such scenario. I will follow this story.

Shin H, & Iwasaki A (2012). A vaccine strategy that protects against genital herpes by establishing local memory T cells. Nature, 491 (7424), 463-467 PMID: 23075848

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The effect of MyD88 deletion on autoimmunity driven by Foxp3 inactivation

Over last 10 years few subjects in immunology have received more attention than regulatory CD4 T cells called in abbreviation Tregs. Tregs are considered to have the potent suppression activity over adaptive immune responses and their lack may result in the autoimmunity development. The central trait pertinent to Tregs is the expression of the transcription factor Foxp3.  The essential role of Foxp3 in Tregs’ life is underscored by the fact that Foxp3-deficient mice or human patients with mutations in the respective gene acquire massive systemic autoimmunity due to the absence of Tregs and generally do not fare well. The publication I have found adds yet another twist to Tregs and Foxp3 story. It turns out that the concurrent to Foxp3 deletion of MyD88 (the crucial adaptor protein linking the innate recognition of microbial signature patterns to the expression of genes involved in defense mechanisms) imparts effects that are not identical between major environmental surfaces (skin, gastrointestinal tract or lungs) and the systemic compartments.

The link: http://www.jci.org/articles/view/40591

Foxp3-deficient mice suffer from the advanced inflammatory skin condition and as a result have grossly increased skin pathology indicators like dryness, loss of hair and bleeding. Apart from that their ears and tails are seriously necrotized. However, animals deleted for both Foxp3 and MyD88 show many substantial improvements. Authors demonstrate that the removal of MyD88 from Foxp3-deficient background diminishes immune infiltration to the epidermis and locally deactivates molecular pathways involved in the amplification of inflammatory signals and cellular trafficking (NF-κB translocation to the nucleus, the expression of ICAM-1 on keratinocytes). Additionally, the skin level of numerous cytokines is reduced in doubly deficient animals compared to mice with the single Foxp3 deletion.

What is remarkable, MyD88 deletion on Foxp3-deficient background has also significant systemic effects because such mice grow to much bigger size than visibly runted Foxp3 single mutants. Therefore investigators analyze the extend of immune infiltration in multiple organs of double Foxp3/MyD88 mutants and find out that they have decreased inflammation scores and the expression of pro-inflammatory cytokines not only in the skin but in the small intestine and lungs as well. However, the alleviating consequences of MyD88 removal are restricted to environmental surfaces as the symptoms characteristic for Foxp3 deletion continue unabated in the liver and the pancreas of Foxp3/MyD88-deficient animals (and are even enhanced in their salivary glands). Moreover, the detailed examination of spleen and lymph nodes (authors indicate that they focus on skin draining lymph nodes and mesenteric lymph nodes) shows that cellular counts, proliferation indicators and the expression of various cytokines are elevated in Foxp3/MyD88-deficient mice compared to animals with Foxp3 deletion.

Such difference between the systemic compartments and environmental surfaces could be explained by several factors. Authors show that introducing MyD88 deletion to Foxp3-deficient background disrupts the chemokine gradient between lymph nodes and effector tissues. They also demonstrate that homing ability of CD4 T cells to lungs is incapacitated and as a consequence lymphocytes may accumulate in draining lymph nodes. Finally, in a series of adoptive transfer experiments it is established that the protective effect of MyD88 deletion acts at the level of target tissue and is independent on whether CD4 T cells express MyD88.

In an interesting, although not entirely conclusive part of the paper authors follow the hypothesis that the protective influence of MyD88 deletion in this model may be due to the removal of capability to process activation signals derived from microbiota. To prove such concept they attempt to mimic the effect of MyD88 ablation by purging commensal bacteria from gastrointestinal tracts of Foxp3-deficient animals with two different antibiotic treatments. The first such treatment includes two antibiotics (doxycycline and cotrimoxazole) and indeed relieves some symptoms of Foxp3 inactivation in the skin and lungs. However, the second regimen comprising four antibiotics (kanamycin, vancomycin, metronidizol, and amphotercin-B) actually worsens the state of Foxp3-deficient animals and accelerates their death. I would be interesting to know what part of commensal microflora can be hold responsible for either such protective or detrimental effects in the context of ongoing autoimmunity.

Rivas MN, Koh YT, Chen A, Nguyen A, Lee YH, Lawson G, & Chatila TA (2012). MyD88 is critically involved in immune tolerance breakdown at environmental interfaces of Foxp3-deficient mice. The Journal of clinical investigation, 122 (5), 1933-47 PMID: 22466646

The different chemokine profile in HIV-exposed seronegative persons

The studies on human population infected with or exposed to HIV have brought the description of several virus-refractory phenotypes. Among them are long-time non-progressors and HIV-exposed seronegative persons. The first group is able to control virus replication without the anti-retroviral therapy, maintain the normal CD4 T cell number and avoid the chronic immune activation that is normally associated with HIV infection. In consequence, long-time non-progressors can live with the presence of HIV for many years with minimal or without any ill effects. The second group is less widely known and comprises people who despite the persistent exposure to the virus do not become infected. In ordinary circumstances HIV gets into the body through mucosal surfaces at genital organs. HIV-exposed seronegative persons (HESN) appear to differ from the virus-sensitive population in terms of chemokine profile at the natural infection site.

The link: http://www.nature.com/mi/journal/v5/n3/abs/mi20127a.html

The study is conducted among Kenyan commercial sex workers divided into three categories. The experimental group includes HESN women, whereas control groups are HIV-1 negative women and HIV-1 infected patients. Authors analyze the cytokine/chemokine profile in the cervicovaginal lavage of each group. They detects that MIG and IP-10 (two IFN-γ inducible chemokines involved in the leukocyte trafficking) and cytokine IL-1α are expressed at lower level in the HESN cohort. Both chemokines are involved in the mucosal migration of activated CD4 T cells – the main HIV target. Hence one of HESN phenotype explanation might be that these people display the state of immune quiescence at their genital mucosa and simply do have enough number of activated CD4 T cells to become infected by virus.

To validate this point investigators examine plasma chemokine/cytokine profile in all studied groups as lymphocytes migrate to mucosal surfaces according to the chemokine gradient between blood and mucosa. They demonstrate that MIG levels in HESN group were higher in systemic compartments than at the genital mucosa whereas the HIV-1 negative population shows the opposite trend. Additionally, HESN subjects uniquely display the decreasing gradient for IP-10 from plasma to mucosal surfaces. The supportive data in this paper include the analysis of CXCR3 (the receptor bound by MIG and IP-10) on CD4 and CD8 T cells collected from the genital mucosa. Authors also study the expression of antiproteases in the genital tract as they are important factors regulating mucosal chemokine levels. It would be interesting to know what really drives the decreased chemokine levels in the genital tract of HESN.

J Lajoie, J Juno, A Burgener, S Rahman, K Mogk, C Wachihi, J Mwanjewe, F A Plummer, J Kimani, T B Ball, and K R Fowke (2012). A distinct cytokine and chemokine profile at the genital mucosa is associated with HIV-1 protection among HIV-exposed seronegative commercial sex workers Mucosal Immunology DOI: 10.1038/mi.2012.7

Degradation of chemokines by food-borne bacterium

The diet, previous or ongoing encounters with infectious organisms and parasites as well as bacterial microflora that inhabit the intestinal tract or other mucosal surfaces – all these factors influence the quality of immune responses. To mention just one relevant case – in developing countries people appear to be less affected by autoimmune diseases but in wealthy societies autoimmunity represents the constantly growing problem. This phenomenon may be partially due to the much lower level of contact with parasitic worms in places where higher civilization level is attained. It has been shown that some parasites (Schistosoma mansoni is the best known example) seem to be able to exert the regulatory effect on mammalian immune responses and thereby reduce the risk of inappropriate reactions to self-antigens. The microflora may also be a factor in autoimmunity development. The publication I have found describes details of interactions between probiotic-associated bacterial molecule and pro-inflammatory chemokines many of which are involved in autoimmune diseases.

The link: http://www.cell.com/cell-host-microbe/retrieve/pii/S1931312812000662

This report is a continuation of previous study where it has been shown that a cell surface protein from Lactobacillus casei strain derived from VSL#3 (a probiotic food product used in management of ulcerative colitis) can degrade IP-10 (interferon gamma induced protein 10, known also as CXCL10 – a pleiotropic pro-inflammatory chemokine). Authors use molecular biology techniques to prove that the described earlier protein is a serine protease. The action of this protease is not specific to IP-10 as it degrades a number of other chemokines (CXCL9, CXCL11, CXCL12, CX3CL1 and CCL11). On the other hand several well-known pro-inflammatory agents like RANTES, IL-6, IFNγ and TNF are not affected.

Investigators attempt to translate L.casei-dependent chemokine degradation into the physiological setting. To this end they use TNFΔARE/+ model (mice lacking post-transcriptional regulation of TNF that develop spontaneous inflammatory bowel disease). The intraperitoneal injections of L.casei-conditioned media reduces several pro-inflammatory parameters like ileal IP-10 level, activation of certain signaling pathways and infiltration of ileum by mononuclear cells or T cells. Authors proceed to screening fecal human samples for bacteria displaying similar abilities and identify microflora-derived L.casei strain that also degrades IP-10. This strain and its mutated version with the disrupted copy of gene encoding the protease in question are used to feed mice with intestinal inflammation (the different disease model is used this time – Rag2-/- mice that received IL-10 deficient CD4 T cells). The presence of L.casei with protease reduces cecal inflammation indices like IP-10 level or T cell influx whereas the protease-deficient strain is unable to influence the above parameters.

Such capacity of L.casei – the regulation of immune responses via degradation of potent pro-inflammatory agents has provoked me to hypothesize about the origin of dairy products consumption. The hypothesis I have is a tentative one and I am not sure whether it reflects the true reason-effect relationship. I am also aware that it drifts far away from the data presented in the discussed paper and I do not know if it has not has been already proposed by somebody else. Let me state few facts: (1) L.casei belongs to the bacterial group called LAB (lactic acid bacteria). LAB comprises species that are associated with mammalian mucosal surfaces and food products including milk (they are not limited to milk, however). (2) The human capacity to consume milk and dairy products beyond infancy period dates back to the time of agricultural revolution and the transition from hunter-gatherer lifestyle. Interestingly, genomic data indicate that the enzyme metabolizing milk lactose has undergone huge selection events which are comparable to the selection that in the recent human history affected factors responsible for skin pigmentation.

What was the true reason behind our ability to digest milk as adults? Could it be the presence of food-borne bacteria that were able to regulate mucosal immune responses? The transition to the agriculture had to involve many dramatic changes in human diet (most probably in human microbiota, too) and first farmers tended to be actually less healthy than hunters-gatherers (I need to indicate that I have no specialist knowledge of ethnography and I rely here on several general science books I have read – most notably Pandora’s Seed: Why the Hunter-Gatherer Holds the Key to Our Survival by Spencer Wells). Might the extended period of milk consumption be able to alleviate the stress on human physiology imparted by modifications to the original human lifestyle?

Such line of thinking brings another question. What could be special about the original human microbiome? The remaining hunter-gatherer people are distinctively free from diseases comprising so-called metabolic syndrome (source: The Cambridge Encyclopedia of Hunters and Gatherers). Might it be partially due to specific microbiota they harbour? The only relevant report I could find indicates that the oral microbiome of Batwa pygmies is significantly more diverse than in their agricultural neighbors (High diversity of the saliva microbiome in Batwa Pygmies: PLoS One. 2011; 6(8):e23352). Maybe we can find solutions to ever-increasing burdens of civilization in those vanishing people?

von Schillde MA, Hörmannsperger G, Weiher M, Alpert CA, Hahne H, Bäuerl C, van Huynegem K, Steidler L, Hrncir T, Pérez-Martínez G, Kuster B, Haller D. (2012). Lactocepin Secreted By Lactobacillus Exerts Anti-Inflammatory Effects By Selectively Degrading Proinflammatory Chemokines Cell Host&Microbe DOI: 10.1016/j.chom.2012.02.006

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