Impact of a fat-rich diet on the pathogenesis of SIV infection Cuiling Xu Center for Vaccine Research University of Pittsburgh

Background Persistent immune activation and inflammation (IA/INFL) are hallmarks of chronic HIV/SIV infection and strong predictors of disease progression. Mucosal damage-resulted microbial translocation (MT) is central to AIDS pathogenesis by inducing persistent immune activation and inflammation. There is little information regarding the impact of environmental factors, such as life style and dietary habits on the integrity of gastrointestinal mucosa.

Background High fat diet (HFD) -> obesity and type 2 diabetes (Kahn et al, 2006), clinical conditions characterized by a chronic state of low-grade systemic inflammation (Wellen & Hotamisligil, 2005) HFD was also shown to directly impair the gut barrier and induce MT, and produce alterations of the gut microbiota (Muller et al, 2015) HFD induced dyslipidemia, infiltration of macrophages to the aorta, and alterations in the coagulation profiles to hypercoagulable status (Yamada et al, 1992; Henry et al, 2009) Therefore, it is conceivable that HFD might impact the natural history of SIV infection

NHP models of SIVsab infection PIGTAILED MACAQUE Non natural host of SIVsab Altered mucosal barrier Microbial translocation Chronic IA/INFL SIV comorbidities (CVD) Progression to AIDS AFRICAN GREEN MONKEY Natural host of SIVsab Maintenance of mucosal barrier Lack of microbial translocation Lack of chronic IA/INFL Lack of comorbidities Lack of disease progression To test this hypothesis, we utilized two nonhuman primate models previously developed in our lab. The well-known pigtail macaques are non-natural host of SIV sab. When infected, they have altered… and eventually progress to AIDS. Whereas, african green monkeys are the natural… when infected, they lack all these pathogenic characteristics and do not progress to AIDS.

Study Design PTMs Fat Diet (N=5), initiated 50 days before inoculation Control (N=10) AGMs Fat Diet (N=4), initiated 102 days before inoculation Control (N=9) Commercially available HFD (~ 40% of calories from fat) In this study, we had a group of five PTMs that started receiving a commercially available high fat diet 50 days before inoculation, with a control group of 10 PTMs. And we also had a group of four AGMs that started receiving the high fat diet 102 days before inoculation, with a control group of 9 AGMs.

No significant difference in viral loads between fat diet and control groups When we are looking at the viral load, we didn’t see significant difference between the fat diet and control groups in either species.

Peripheral CD4 level showed a mild decrease with fat diet prior to and after infection - Fat diet initiation - Infection However, looking at their CD4 levels, here you can see, the start of the yellow block marks the initiation of fat diet, and start of the pink block marks virus inoculation. We saw a mild decrease of peripheral CD4 with fat diet before and after infection.

Gut CD4 decreased in AGMs receiving fat diet prior to and after infection When we are looking at gut CD4 levels, we saw a much bigger decrease in the AGMs with fat diet prior to infection and persist until after infection. However, we did not see a big difference in PTMs between fat diet and control group. This might be due to viral infection already causing very severe damage in the gut of PTMs, therefore masking the effect of fat diet.

Elevated immune activation in AGMs receiving fat diet This decrease in CD4 level could indicate a high level of immune activation and inflammation in these fat diet receiving AGMs. So then we looked at these markers, and we did see increased HLA-DR CD38 and Ki-67 on both CD4 and CD8 T cells from these AGMs. Not much difference was seen in PTMs, again probably because they already have highly increased immune activation and inflammation due to the infection, so it’s hard to see the effect of dietary factor.

Elevated inflammatory markers in AGMs receiving fat diet Similarly, we saw increased inflammation in AGMs with fat diet, such as CRP, IP-10, IL-1b and interferon gamma.

Altered chemokine and growth factor profile in AGMs receiving fat diet And they also had altered chemokine and growth factor profile, such as increased RANTES, HGF, VEGF, and MIF.

LPS level increased with fat diet and persist high levels in AGMs after infection It was known that microbial translocation is one of the major contributors to immune activation and inflammation, therefore, we next looked at the microbial translocation maker, LPS. Again, we did not see big difference in PTMs, but in accordance with the CD4 and immune activation and inflammation markers, we did see increased LPS with fat diet before and after infection in AGMs.

Microbiota is significantly altered by fat diet Principal coordinates analysis showing differential clustering between species, and between Fat vs. Non-fat diet groups within species. Interestingly, not only did the fat diet caused gut damage, it also significantly altered the microbiota of these animals. Here in this principal coordinates analysis, you can see AGMs and PTMs cluster very differently, indicating the significant difference between their microbiota profile. Similarly, you can see on the two panels on the right that within each species, fat diet resulted in a significant change of their microbiota. We are currently analyzing these data further to try to identify the microorganisms that dominate these changes, and potentially figure out the drivers for immune activation and inflammation.

Altered blood chemistry and metabolism profile in both AGMs and PTMs receiving fat diet Although in the previously mentioned markers we didn’t see much difference in PTMs, we did see alterations in blood chemistry and metabolism profiles in both species that are receiving fat diet. Therefore, we turned gear towards liver-related pathogenesis to see if we can identify any difference induced by fat diet in PTMs.

Liver steatosis and fibrosis observed in PTMs receiving fat diet And interestingly, in the liver of PTMs receiving fat diet, we did observe steatosis, showing here in the top panels that there is obvious fat infiltration. Some of them even had very severe steatosis. We also saw liver fibrosis showing in the bottom panels that in pre-fat diet liver, collagen is only prominent around portal area and well-organized, whereas in post-fat diet liver, collagen becomes more diffused and formed sinusoidal fibrosis.

Elevated liver fibrosis marker in PTMs receiving fat diet Hyaluronic acid level in blood is a marker associated with development of liver fibrosis. Along with the histological changes, we also observed increased hyaluronic acid, which is a liver fibrosis marker, in the blood stream of fat diet PTMs, consistent with the trichrome staining that you just saw.

Major impact of fat diet on disease progression Most importantly, fat diet had a major impact on the survival of these animals, potentially due to the combined effect of all the previously mentioned pathogenesis-related modifications. Here you can see the survival of the fat diet group PTMs was significantly impacted by fat diet, and even in AGMs, one of the fat diet receiving animals died in spite of their nonpathogenic nature of SIV infection. Notably, in the AGM Fat Diet group (N=4), one of the AGMs died in spite of the nonpathogenic nature of SIV infection in AGMs

Conclusions HFD induced microbial translocation and elevated immune activation and inflammation in nonprogressive models that normally lack these features during infection While the impact on critical parameters of SIV infection was relatively mitigated (especially in PTMs, in which the effect of fat diet could be masked by the infection), HFD greatly impacted survival in both species HFD caused altered metabolism profile and liver modifications in both species HFD, especially when associated with other environmental risk factors, could impact SIV pathogenesis and disease progression With these evidence, we concluded that…

Acknowledgements Apetrei/Pandrea lab University of Colarado Tianyu He Jenny Stock Benjamin Policicchio Dongzhu Ma George Haret-Richter Tammy Dunsmore Kathryn Martin University of Colarado Daniel Franck Cara Wilson University of Vermont Russell Tracy Rush University Alan Landay Funding RO1 HL117715 (IP) and RO1 HL123096 (IP) from the NHLBI