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The early cost of HIV....in the SIV gut....priobiotics: "Early Mucosal Sensing of SIV Infection by Paneth Cells Induces IL-1ß Production and Initiates Gut Epithelial Disruption"
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Inflammatory response breaks down intestinal lining, but help may come from friendly bacteria
August 29, 2014
(SACRAMENTO, Calif.) -http://www.ucdmc.ucdavis.edu/publish/news/newsroom/9205
Researchers at UC Davis have made some surprising discoveries about the body's initial responses to HIV infection. Studying simian immunodeficiency virus (SIV), the team found that specialized cells in the intestine called Paneth cells are early responders to viral invasion and are the source of gut inflammation by producing a cytokine called interleukin-1 beta (IL-1ß).
Though aimed at the presence of virus, IL-1ß causes breakdown of the gut epithelium that provides a barrier to protect the body against pathogens. Importantly, this occurs prior to the wide spread viral infection and immune cell killing. But in an interesting twist, a beneficial bacterium, Lactobacillus plantarum, helps mitigate the virus-induced inflammatory response and protects gut epithelial barrier. The study was published in the journal PLoS Pathogens.
One of the biggest obstacles to complete viral eradication and immune recovery is the stable HIV reservoir in the gut. There is very little information about the early viral invasion and the establishment of the gut reservoir.
"We want to understand what enables the virus to invade the gut, cause inflammation and kill the immune cells," said Satya Dandekar, lead author of the study and chair of the Department of Medical Microbiology and Immunology at UC Davis.
"Our study has identified Paneth cells as initial virus sensors in the gut that may induce early gut inflammation, cause tissue damage and help spread the viral infection. Our findings provide potential targets and new biomarkers for intervening or blocking early spread of viral infection," she said.
In the study, the researchers detected a very small number of SIV infected cells in the gut within initial 2.5 days of viral infection; however, the inflammatory response to the virus was playing havoc with the gut lining. IL-1ß was reducing the production of tight-junction proteins, which are crucial to making the intestinal barrier impermeable to pathogens. As a result, the normally cohesive barrier was breaking down.
Digging deeper, the researchers found the inflammatory response through IL-1ß production was initiated in Paneth cells, which are known to protect the intestinal stem cells to replenish the epithelial lining. This is the first report of Paneth cell sensing of SIV infection and IL-1ß production that links to gut epithelial damage during early viral invasion. In turn, the epithelial breakdown underscores that there's more to the immune response than immune cells.
"The epithelium is more than a physical barrier," said first author Lauren Hirao. "It's providing support to immune cells in their defense against viruses and bacteria."
The researchers found that addition of a specific probiotic strain, Lactobacillus plantarum, to the gut reversed the damage by rapidly reducing IL-1ß, resolving inflammation, and accelerating repair within hours. The study points to interesting possibilities of harnessing synergistic host-microbe interactions to intervene early viral spread and gut inflammation and to mitigate intestinal complications associated with HIV infection.
"Understanding the players in the immune response will be important to develop new therapies," said Hirao. "Seeing how these events play out can help us find the most opportune moments to intervene."
Other UC Davis researchers included: Irina Grishina; Olivier Bourry, William K Hu, Monsicha Somrit, Sumathi Sankaran-Walters, Chris A Gaulke, Anne N Fenton, Jay A Li, Robert W.Crawford, Frank Chuang, Ross Tarara, Maria Marco, Andreas J Baumler, Holland Cheng and Satya Dandekar.
The study was funded by: the NIAID-NIH, UC Davis RISE, California HIV Research Program, and NIH BIRCWH.
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IAC/2014-Melbourne: Probiotic and IL-21 Treatment Promotes Th17 Cell Recovery in ARV-Treatment of Pigtail Macaques - (08/06/14)
Editorial - Probiotic Safety and Risk Factors.......Journal of Clinical Gastroenterology, May/June 2013, Floch, Martin H. MD, MACG, AGAF Department of Internal Medicine, Section of Digestive Diseases, Yale University School of Medicine, New Haven, CT.......http://www.natap.org/2013/HIV/060413_01.htm
Lactobacillus Bacteremia Associated With Probiotic Use in a Pediatric Patient With Ulcerative Colitis.......Case Report, Journal of Clinical Gastroenterology
May/June 2013......http://www.natap.org/2013/HIV/060513_03.htm
CROI/2012: Probiotic Supplementation of ARV Treatment during SIV Infection of Pigtail Macaques Results in Enhanced GI Tract CD4+ T Cell Frequency and Immunological Function ........http://www.natap.org/2012/CROI/croi_112.htm
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"In the present study, we investigated the earliest effects of SIV, prior to acute mucosal CD4+ T cell depletion, on epithelial barrier integrity and mucosal immune response to pathogenic (Salmonella enterica serovar Typhimurium, S. Typhimurium) and non-pathogenic (Lactobacillus plantarum, L. plantarum) bacteria in vivo. Our findings showed that the gut epithelium was the initial target of viral pathogenesis, as evidenced by impaired expression and disorganization of epithelial tight junction proteins, which were correlated to increased expression of interleukin-1ß (IL-1ß). We identified Paneth cells as the dominant source of the early innate IL-1ß immune response. At this time-point, no defects in mucosal immune response to either pathogenic or commensal bacteria were observed. In fact, mucosal exposure to L. plantarum rapidly dampened SIV-induced inflammation through the inhibition of the NF-κB pathway. Our study identified, for the first time, Paneth cells as an initial source of gut inflammation and IL-1ß signaling during early viral infection. In addition, anti-inflammatory and epithelial repair effects of L. plantarum suggest the potential role of commensal bacteria in reversing the early effects of viral pathogenesis.......The ability of L. plantarum to modulate NF-κB activation and ameliorate epithelial defects makes it an attractive therapeutic adjuvant. These results highlight the importance of the trialogue between the epithelium, immune cells, and commensal organisms in the restoration and protection of the intestinal mucosa [51]. By further understanding the mechanisms that underlie the host/microbiota relationship in health and HIV disease, we can capitalize on their evolved synergy while identifying gaps in mucosal defenses that can be fortified through therapy.......We found that L. plantarum rapidly induced intestinal epithelial repair in SIV infected macaques through anti-inflammatory effects that were evident by decreased expression of IL-1ß and inflammatory chemokines. Previous studies reported on the ability of Lactobacillus species to enhance epithelial barrier integrity via tight junction regulation [44]-[46]. Lactobacilli are known to regulate the NF-κB signaling cascade in both intestinal epithelial and antigen presenting cells [47], [48]. In the current study, significant correlations were found linking disruption of epithelial tight junctions, induction of IL-1ß levels, NF-κB activation and the ability of L. plantarum to downregulate these pathologic processes. This raises a possibility of exploiting of L. plantarum to intervene the early mucosal-viral interactions that may influence gut inflammation. In addition to its anti-inflammatory effects, we observed enhanced recruitment of Th17 cells in response to L. plantarum, mostly likely due to the induction of CCL20 expression. This recruitment of Th17 cells may have a role in epithelial repair. Our findings suggest a supportive role of L. plantarum in overcoming SIV-induced gut inflammation and epithelial tight junction disruption. However, unintended consequences of an L. plantarum probiotic therapeutic adjuvant may include increased viral replication through recruitment of virus-susceptible Th17 cell targets and viral dissemination through the induction of the CXCR4-CXCL12 axis. Our findings raise an important consideration in the development of probiotic therapies for HIV infection and highlight the need for a better characterization of probiotic bacterial functions and effects [49], [50]."
Early Mucosal Sensing of SIV Infection by Paneth Cells Induces IL-1ß Production and Initiates Gut Epithelial Disruption
Abstract
HIV causes rapid CD4+ T cell depletion in the gut mucosa, resulting in immune deficiency and defects in the intestinal epithelial barrier. Breakdown in gut barrier integrity is linked to chronic inflammation and disease progression. However, the early effects of HIV on the gut epithelium, prior to the CD4+ T cell depletion, are not known. Further, the impact of early viral infection on mucosal responses to pathogenic and commensal microbes has not been investigated. We utilized the SIV model of AIDS to assess the earliest host-virus interactions and mechanisms of inflammation and dysfunction in the gut, prior to CD4+ T cell depletion. An intestinal loop model was used to interrogate the effects of SIV infection on gut mucosal immune sensing and response to pathogens and commensal bacteria in vivo. At 2.5 days post-SIV infection, low viral loads were detected in peripheral blood and gut mucosa without CD4+ T cell loss. However, immunohistological analysis revealed the disruption of the gut epithelium manifested by decreased expression and mislocalization of tight junction proteins. Correlating with epithelial disruption was a significant induction of IL-1ß expression by Paneth cells, which were in close proximity to SIV-infected cells in the intestinal crypts. The IL-1ß response preceded the induction of the antiviral interferon response. Despite the disruption of the gut epithelium, no aberrant responses to pathogenic or commensal bacteria were observed. In fact, inoculation of commensal Lactobacillus plantarum in intestinal loops led to rapid anti-inflammatory response and epithelial tight junction repair in SIV infected macaques. Thus, intestinal Paneth cells are the earliest responders to viral infection and induce gut inflammation through IL-1ß signaling. Reversal of the IL-1ß induced gut epithelial damage by Lactobacillus plantarumsuggests synergistic host-commensal interactions during early viral infection and identify these mechanisms as potential targets for therapeutic intervention.
Author Summary
The loss of intestinal CD4+ T cells in chronic HIV infection is associated with impaired immune responses to pathogens, aberrant immune activation, and defects in the gut epithelial barrier. While much is known about the pathogenesis of HIV in chronic disease, less is known about the defects that occur prior to gut CD4+ T cell depletion and whether these defects alter host interactions with pathogenic and commensal bacteria. Using a non-human primate model of HIV infection, we examined the immune and structural changes in the gastrointestinal tract 2.5 days following SIV infection. Paneth cells, in immediate proximity of SIV infected immune cells, generated a robust IL-1ß response. This IL-1ß response correlated with defects in epithelial tight junctions and preceded the IFN-α response, which is characteristic of innate antiviral immune responses. Despite this inflammatory environment, we did not observe defects in mucosal immune responses to pathogenic or commensal bacteria. In fact, commensal bacteria were able to dampen the IL-1ß response and ameliorate tight junction defects. Our study highlights the importance of the gut epithelium in HIV infection, not just as a target of pathogenesis but also the initiator of immune responses to viral infection, which can be strongly influenced by commensal bacteria.
Introduction
Chronic inflammation and disease progression in HIV infection is attributed to dysfunction in the structure of the intestinal epithelial barrier as well as impairment of the mucosal immune response resulting in increased microbial translocation [1]-[3], dysbiosis of the gut microbiome [4]-[6], and enteric opportunistic infections [7]. Incomplete recovery of gut homeostasis, despite antiretroviral therapy, contributes to the persistence of immune activation in HIV infected patients [8]-[10]. Studies in HIV infected patients and SIV infected non-human primates have shown massive dissemination of viral infection in the gut mucosa during the primary acute stage of infection leading to severe and rapid CD4+ T cell depletion [11]-[14], which persists through all stages of infection [15], [16]. In contrast, CD4+ T cell loss is progressive in peripheral blood and lymph nodes. Loss of mucosal Th17 CD4+ T cell subset coincides with epithelial barrier disruption and is linked to increased microbial translocation and chronic immune activation [17], [18]. Although immune dysfunction following mucosal CD4+ T cell loss is well described, it is not known whether HIV can alter mucosal function and epithelial integrity prior to and independent of CD4+ T cell depletion in vivo. Further, our understanding of mucosal resident cells that are early responders to the virus and their inflammatory signaling networks is limited.
The intestinal epithelium is functionally diverse. In addition to the digestive and absorptive functions, it plays a critical role in microbial sensing and innate antimicrobial response [19]. Secretory lineages of the intestinal epithelium produce antimicrobial products such as mucins by Goblet cells and defensins and inflammatory cytokines by Paneth cells [20]. Expansion of Paneth cells during chronic SIV infection has highlighted its important role in imparting innate defense in gut mucosa during chronic SIV infection [21]. Although the Paneth cell response to microbial pathogens is well investigated, there is no information about their response to pathogens during early HIV and SIV infections and viral pathogenesis.
There is increasing evidence that viral infections can alter the host-commensal relationship [22]. HIV and SIV induced changes in the gut microenvironment may have a profound effect on the mucosal response to incoming enteric pathogens as well as local commensal bacteria. To assess the early changes in mucosal responses induced by SIV infection, use of an in vivo intestinal model is essential, as in vitro cell culture studies fail to replicate the complex cellular interactions and anaerobic microenvironment of the gut. We developed the simian ligated intestinal loop model, which most closely recapitulates the anaerobic gut microenvironment. By directly injecting bacteria into the intestinal lumen, this model facilitates the capture of the in vivo dynamics between microbes, the gut epithelium, and immune cell populations during the viral infection [17].
In the present study, we investigated the earliest effects of SIV, prior to acute mucosal CD4+ T cell depletion, on epithelial barrier integrity and mucosal immune response to pathogenic (Salmonella enterica serovar Typhimurium, S. Typhimurium) and non-pathogenic (Lactobacillus plantarum, L. plantarum) bacteria in vivo. Our findings showed that the gut epithelium was the initial target of viral pathogenesis, as evidenced by impaired expression and disorganization of epithelial tight junction proteins, which were correlated to increased expression of interleukin-1ß (IL-1ß). We identified Paneth cells as the dominant source of the early innate IL-1ß immune response. At this time-point, no defects in mucosal immune response to either pathogenic or commensal bacteria were observed. In fact, mucosal exposure to L. plantarum rapidly dampened SIV-induced inflammation through the inhibition of the NF-κB pathway. Our study identified, for the first time, Paneth cells as an initial source of gut inflammation and IL-1ß signaling during early viral infection. In addition, anti-inflammatory and epithelial repair effects of L. plantarum suggest the potential role of commensal bacteria in reversing the early effects of viral pathogenesis.
Discussion
Our study, for the first time, reports that Paneth cells are early sensors of virally infected immune cells in the intestinal mucosa. Their inflammatory response is mediated through robust IL-1ß signaling, with profound implications on early tissue damage. Thus, Paneth cells play a critical role in the induction of gut inflammation during the early stages of viral infection, prior to the depletion of CD4+ T cells. To our knowledge, this is the first description of IL-1ß production by Paneth cells.
While the mechanism by which Paneth cells sense and respond to pathogenic bacteria is well characterized, our understanding of their response to HIV infection is limited [20]. We found that SIV infected cells were localized in close proximity of the crypt epithelium, potentially exposing Paneth cells to viral antigens or inflammatory cytokines released by the infected cells. In HIV infection, virus has been shown to induce NLRP3-inflammasome expression and IL-1ß production in myeloid cells [27]. Though we cannot definitively attribute the induction of IL-1ß to a specific stimulus, NLRP3 expression was increased in the gut mucosa suggesting potential involvement of an NLRP3-inflammasome mediated pathway in Paneth cells during SIV infection. Our findings highlight the need for future investigations to determine the mechanisms of Paneth cell sensing and response to viral infections and their role in the induction of host innate response to HIV.
We previously reported an increased expression of enteric defensins in Paneth cells during primary and chronic SIV infection that correlated with viral loads [21]. The loss of defensin accumulation in these cells correlated with disease progression and opportunistic infections. In the present study, we did not observe an increase in enteric defensin gene expression at 2.5 days of SIV infection. This suggests that the IL-1ß response precedes the upregulation of defensin expression in Paneth cells. Similarly, we did not detect a significant increase in the expression of IFN-α or IFN stimulated genes (ISG). The type 1 IFN response is critical in the early containment of viral replication [28]. However, this involves the recruitment of plasmacytoid dendritic cells to the gut mucosa and may require higher levels of viral replication than occurs at 2.5 days following SIV infection [28]-[30]. Thus, IL-1ß production by Paneth cells represents a local response to SIV infection at a time point when viral presence is low in the intestinal mucosa, and may critically impact innate immune cell subsets such as macrophages and innate lymphoid cells (ILC), which express IL-1ß receptors [31], [32].
Inflammatory cytokines have been shown to disrupt epithelial barrier integrity [33]. Exposure to IL-1ß increased permeability in intestinal epithelial cell cultures by decreasing epithelial tight junction protein expression [34]-[36]. Increased IL-1ß expression at 2.5 days of SIV infection negatively correlations with expression of tight junction components in our study, suggesting that IL-1ß initiates intestinal epithelial barrier defects. Other inflammatory cytokines, such as IFN-γ and TNF-α, have also been shown to cause disruption of epithelial cell tight junctions in vitro [37]. However, we did not detect an upregulation of IFN-γ or TNF-α expression by transcriptome analysis in vivo, suggesting that these cytokines might not contribute significantly towards intestinal epithelial changes during early infection. HIV envelope protein gp120 was shown to induce defects in epithelial tight junctions, only when added apically to epithelial cell cultures. No effects were observed when gp120 was added basolaterally [38]. This mechanism is unlikely to play a role in epithelial integrity defects observed in our study, given that the few SIV infected immune cells detected were localized to the basolateral side of the intestinal epithelium.
In chronic SIV disease, epithelial barrier disruption has been shown to lead to increased microbial translocation. However, the changes in the intestinal epithelial barrier that occur during early viral infection did not result in systemic dissemination of bacteria and microbial products. This discrepancy is likely due to the preservation of mucosal CD4+ T cells in early infection, as our previous study had shown that the depletion of Th17 cells, in chronic SIV infection, results in the increased dissemination of pathogenic S. Typhimurium [17].
The ability of the mucosal immune system to rapidly eradicate pathogens while maintaining tolerance to commensal bacteria is critical to the maintenance of intestinal homeostasis. The occurrence of aberrant host immune responses to commensal bacteria has been reported during chronic inflammatory conditions such as inflammatory bowel diseases (IBD) [39] and recently in acute Toxoplasma gondii infection [22]. Aberrant inflammatory response to commensal bacteria by peripheral monocytes of individuals with chronic HIV infection has been reported [40]. It is not known whether acute HIV infection might obfuscate the host's ability to distinguish between pathogen and commensals. Aberrant immune responses to commensal bacteria during chronic HIV infection may be attributed to increased microbial translocation [2], immune activation of antigen presenting cells [40], [41], and increased TLR2 and TLR4 expression [42], [43]. However, there have been no known studies that have interrogated gut mucosal immune responses to commensal bacteria in the context of early HIV infection. In our study, SIV infected animals had enhanced inflammatory responses to S. Typhimurium, compared to SIV-negative controls, but showed no significant changes in the response to L. plantarum. Thus, in early SIV infection, the host maintains its ability to distinguish pathogenic and commensal bacteria and mount the proper immune response.
We found that L. plantarum rapidly induced intestinal epithelial repair in SIV infected macaques through anti-inflammatory effects that were evident by decreased expression of IL-1ß and inflammatory chemokines. Previous studies reported on the ability of Lactobacillus species to enhance epithelial barrier integrity via tight junction regulation [44]-[46]. Lactobacilli are known to regulate the NF-κB signaling cascade in both intestinal epithelial and antigen presenting cells [47], [48]. In the current study, significant correlations were found linking disruption of epithelial tight junctions, induction of IL-1ß levels, NF-κB activation and the ability of L. plantarum to downregulate these pathologic processes. This raises a possibility of exploiting of L. plantarum to intervene the early mucosal-viral interactions that may influence gut inflammation. In addition to its anti-inflammatory effects, we observed enhanced recruitment of Th17 cells in response to L. plantarum, mostly likely due to the induction of CCL20 expression. This recruitment of Th17 cells may have a role in epithelial repair. Our findings suggest a supportive role of L. plantarum in overcoming SIV-induced gut inflammation and epithelial tight junction disruption. However, unintended consequences of an L. plantarum probiotic therapeutic adjuvant may include increased viral replication through recruitment of virus-susceptible Th17 cell targets and viral dissemination through the induction of the CXCR4-CXCL12 axis. Our findings raise an important consideration in the development of probiotic therapies for HIV infection and highlight the need for a better characterization of probiotic bacterial functions and effects [49], [50].
In summary, our study has identified the gut epithelium, specifically Paneth cells, as a site of sensing and response of viral infection and an inducer of gut inflammation through IL-1ß signaling during early SIV infection. The ability of L. plantarum to modulate NF-κB activation and ameliorate epithelial defects makes it an attractive therapeutic adjuvant. These results highlight the importance of the trialogue between the epithelium, immune cells, and commensal organisms in the restoration and protection of the intestinal mucosa [51]. By further understanding the mechanisms that underlie the host/microbiota relationship in health and HIV disease, we can capitalize on their evolved synergy while identifying gaps in mucosal defenses that can be fortified through therapy.
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