Antiviral therapy (ART) could significantly inhibit the human immunodeficiency virus (HIV). However, even if under ART and sustained virological suppression, a certain portion of HIV-infected individuals still cannot achieve complete immune reconstitution (1–3), they are called immune non-responders. The immune non-responders are prone to opportunistic infections and usually have a poor prognosis. Apart from acquired immunodeficiency syndrome (AIDS) -related diseases and deaths, the probability of non-AIDS-related diseases and deaths are higher for those with immune non-response (4–6).

Recent theoretical developments have revealed that the potential intermediate pathway for excessive early mortality in HIV-infected immunological non-responders due to malnutrition (7). Thus, nutrition plays an important role in the immune reconstitution among patients with HIV/AIDS. Besides direct damage of HIV to the immune system, HIV-positive individuals are prone to malnutrition due to inadequate dietary intake, nutritional losses, metabolic changes, and increased demand for macronutrients and micronutrients (8, 9). The integrity of the intestinal barrier is essential for human absorption of nutrients and health. Persistent intestinal mucosal barrier damage affects nutrient absorption (10), thereby leading to malnutrition. In addition, malnutrition also affects the integrity of the intestinal barrier and the balance of intestinal microbes (11, 12), further hindering the reconstruction of immune function.

Considering the important role of nutrition in immunity, a large number of studies have made efforts to improve the nutritional status of AIDS patients. Studies have shown that enteral nutritional supplementation is beneficial for the improvement of nutritional status and the recovery of CD4⁺ T cells (13–18). In addition, the composition of nutrition supplements, patient characteristics, and treatment methods vary greatly in different intervention studies. However, there is still a lack of relevant research on nutritional intervention for patients with immunological non-response.

Here, we proposed a pre-digested enteral nutritional supplement and evaluated its clinical effects on AIDS patients with immunological non-response by detecting serum levels of immune indicators and pro-inflammatory cytokines. Meanwhile, we also evaluated the effects of the pre-digested nutritional supplement on the intestinal barrier function and nutritional status in these patients.

In this nutritional intervention trial, we showed that the WBC, neutrophils, and CD4⁺ T cells increased significantly after three months of nutritional intervention. Overall, the nutritional status of the patients was significantly improved as well. In terms of cytokines, serum levels of IL-1β in patients after nutritional intervention decreased significantly. However, we observed that the TNF-β level increased significantly after nutritional intervention. In addition, serum levels of DAO, D-lactate, and LPS were significantly decreased in patients undergoing nutritional intervention.

CD4⁺ T cell count and CD4/CD8 ratio serve as a valid predictor of immune reconstitution prognosis after ART (20, 21). Studies have shown that there is a significant correlation between the recovery of CD4⁺ T cells and malnutrition in AIDS patients with poor immune reconstitution after ART (22–25). Malnutrition and HIV play a synergistic role in reducing the number of CD4⁺ T and CD8⁺ T cells (26), delaying skin sensitivity, reducing bactericidal performance (27), and impairing serum immunological response.

In our study, we found that after the nutritional intervention, WBC, neutrophils, and CD4⁺ T cell counts were significantly increased, while CD4/CD8 ratio did not change significantly with showing an increasing trend, suggesting that pre-digested enteral nutritional supplement could help the recovery of CD4⁺ T cells in patients with poor immune reconstitution. Ezeamama et al. has shown that HIV/AIDS patients receiving ART could benefit from nutritional support, especially for the recovery of CD4⁺ T cells (28). Pereira Da Silva et al. found that nutritional intervention could improve the nutritional status of HIV/AIDS patients by increasing the number of lymphocytes (29). A retrospective study also found a 4.7% increase in CD4⁺ T cell counts in patients after the use of nutritional supplements (30). All the above data indicated that adequate nutrition contributes to the reconstitution of immune function in HIV/AIDS patients.

This study also found that CD8⁺ T cells in the peripheral blood of patients with poor immune reconstitution remained elevated after nutritional treatment, which may be the result of persistent immune activation and inflammatory response caused by microbial translocation (31). In addition, CD8⁺ T cell counts increased, possibly because CD8⁺ T cells responded to both viral and intestinal CD4⁺ T cell depletion, and low CD4/CD8 ratio (< 1), which was not altered by nutritional intervention, promoted an increase in CD8⁺ T cell counts (32).

ART could significantly reduce systemic inflammation and immune activation, but it cannot reach the level synchronized with the HIV-uninfected population (33). Especially in patients with poor immune function reconstruction, immune activation and chronic inflammation are persistent, which may be due to malnutrition (34). Malnutrition promotes the phenotypic transformation of immune cells to pro-inflammatory phenotypes (35, 36), secreting pro-inflammatory cytokines such as IL-1β, IL-6, IL-8, TNF-α, and IL-2, while anti-inflammatory cytokines such as IL-1 receptor antagonists, IL-4, IL-10, and IL-13 decrease (37). Malnutrition is more prevalent in AIDS patients with poor immune reconstitution, which may mean that the chronic inflammatory state of patients with poor immune reconstitution is more affected by malnutrition.

High expression of IL-1β is related to HIV replication, at the same time, HIV infection could induce CD4⁺ T cell death in lymphoid tissues through caspase-1-mediated apoptosis and releasing cytokines such as IL-1β and IL-18 (38, 39). Therefore, the sustained high expression of IL-1β may be one of the reasons for poor immune reconstruction in AIDS patients. Through nutritional therapy, the level of IL-1β was significantly reduced, suggesting that nutritional intervention could reduce the inflammatory response by inhibiting HIV replication, and is beneficial in inhibiting HIV-induced CD4⁺ T cell death.

An earlier study has identified the involvement of TNF and IL-1 in HIV viral replication and pathogenesis (40). A prospective cohort study showed that TNF-α production increased in HIV patients receiving ART and increased as the disease progressed (41). There were no significant changes in TNF-α and IL-1α in enteral nutritional treatment except that IL-1β was significantly decreased, which may be due to persistent viral replication in the viral reservoirs of patients with poor immune reconstitution. In addition, TNF-β could activate macrophages and is responsible for cell-mediated immune and phagocyte-dependent protective responses (42). TNF-β is also recognized as a mediator of antigen-induced cell death (AICD) in HIV infection and stimulates HIV replication in T cells and monocyte-derived macrophages (MDM) (43). In our experiments, it is not clear which of these two conditions is responsible for the sustained rise of TNF-β, and further experiments are needed to explore.

The pathogenesis of malnutrition in HIV/AIDS patients is multifactorial, which is mainly related to changes in caloric intake, intestinal injury, nutrient absorption, and increased metabolic demand caused by active infection (8, 44). Among the many factors that cause malnutrition, impairment of intestinal function is associated with immune activation and persistent inflammatory state in AIDS/HIV patients. Damage to the mucosal barrier, microbial translocation, and immune activation during HIV infection may persist even in patients who have successfully received antiretroviral therapy (45). Intestinal barrier dysfunction could lead to increased inflammation, which further impairs the intestinal barrier by disrupting tight junction proteins. Disruption of the intestinal barrier is associated with inflammation of adipose tissue in different disease states, such as obesity, diabetes, HIV infection, and inflammatory bowel disease (46). Therefore, repairing the intestinal barrier after the nutritional intervention may be more conducive to the absorption of nutrients and the recovery of immune function and alleviate chronic inflammation.

After three months of nutritional intervention, we found that the levels of DAO, D-lactate, and LPS were significantly decreased in HIV/AIDS patients with immune non-response, suggesting that the intestinal barrier damage was repaired. The nutrients we used were supplemented with both fish oil and glutamine. Previous studies have also shown that the use of fish oil and glutamine supplements in HIV-infected individuals not only improves muscle protein consumption atrophy but also reduces inflammation and intestinal permeability (47, 48).

The levels of circulating LPS, DAO, and other intestinal barrier damage markers were significantly increased in HIV-infected patients compared with the uninfected control group (49–51). These changes lead to an increase in pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6, thereby triggering an inflammatory response (52), which is sustained by further infiltration of immune cells (e.g. neutrophils and monocytes) in response to this pro-inflammatory environment (53). Therefore, we analyzed the correlation between intestinal barrier damage and inflammatory cytokines and T cells. The results showed that CD4⁺ T cell counts were significantly negatively correlated with the levels of DAO and D-lactate, and the IL-1β level was significantly positively correlated with the serum levels of DAO, D-lactate, and LPS. This suggests that persistent intestinal barrier damage affects immune reconstitution in HIV/AIDS patients, and nutritional intervention could improve this situation.

Malnutrition is still a challenge in AIDS patients. A recent survey found that 21% of AIDS patients had mild to moderate malnutrition and 6% had severe malnutrition, and malnutrition was associated with immune non-response (25). This suggests that nutritional interventions for this population are crucial. Our data show that the weight, BMI, and Hb of patients increased significantly after enteral nutritional treatment, suggesting that nutrition intervention is beneficial for correcting the anemia of patients. Although ALB, PA, and TP did not change statistically, they all showed increasing trends, suggesting that nutritional treatment helps improve the malnutrition status of poor immune reconstitution patients. Studies on nutritional therapy for HIV/AIDS patients (13–18, 54) showed that the Hb and nutritional status of patients with nutritional therapy based on ART were improved. This is consistent with our experimental results.

In healthy people, water accounts for 50% to 70% of the body weight. It transports ingested nutrients into cells and discharges wastes out of the body (55). ICW, ECW, TBW, and ECW/TBW reflect the distribution of water in body cells. The ECW/TBW of healthy people can be maintained at a constant value (EBW/TBW = 0.38) (55, 56). After three months of enteral nutritional treatment, the distribution of intracellular and extracellular water changed, ECW/TBW increased significantly and reached normal value, suggesting that enteral nutritional treatment could help to restore the balance of intracellular and extracellular water to a certain extent, which promotes the transport of nutrients and the discharge of metabolic wastes and provides favorable conditions for the survival of cells in the body. Normally, the TBW/FFM value is 73%, representing 73% of the bodyweight without fat is body water. In our study, TBW/FFM decreased significantly after the nutritional intervention and was closer to the normal value, suggesting that the water content of defatted body weight tended to be more balanced after the nutritional intervention.

Studies have found that in addition to malnutrition due to HIV infection itself, AIDS patients also have ART-related lipodystrophy due to side effects of antiretroviral drugs (8, 57). Our study found that patients with poor immune reconstitution had lower fat and PBF, and the fat content increased significantly after nutritional intervention, while the change of PBF was not obvious. PBF and fat were positively correlated with the incidence of metabolic diseases such as diabetes, hypertension, fatty liver, cardiovascular and other diseases (58). Lower fat and PBF will lead to reduced skin insulation function, hormone disorder, and easy to lead to low immunity (59). The increase of fat content after nutritional intervention not only helps to improve the nutritional status of patients, but also could improve the immunity of patients.

However, our study found that after three months of nutritional intervention, the muscle-related indicators FFM, SLM, SMM, and BCM did not increase significantly, while SMI decreased. A previous study also showed that patients’ nutritional status improved and their fat increased after nutritional intervention, but their muscle mass did not increase, which is consistent with our results (29), suggesting that nutritional intervention alone may have a limited effect on muscle growth.

Taken together, in our study, we explored the clinical effects of a pre-digested enteral nutritional supplement in HIV-infected immunological non-responders. We found that after three months of pre-digested enteral nutritional supplementation, the nutritional status of patients was improved, and importantly, CD4⁺ T cell counts were significantly increased. At the same time, it also has a certain inhibitory effect on chronic inflammation. Further analysis showed that nutritional therapy could repair the damaged intestinal barrier, and the repair of the intestinal barrier was related to the recovery of CD4⁺ T cells and the lower level of IL-1β. The pre-digested enteral nutritional supplement is beneficial to HIV-infected INRs.

The dataset used in the article is available from the corresponding authors upon reasonable request.

The study protocol was approved by the Human Clinical Research Ethics Committee of the First Affiliated Hospital of Kunming Medical University (No. 2018-L-43). Informed consent was obtained from all patients or their relatives or legally accepted representatives.

Y-QK and K-HW conceived and designed the study. S-TG, Y-XW and ZZ performed the experiments. S-TG, J-BZ, Y-XW, YX, ZZ and JG collected the data, S-TG, J-BZ, Y-XW, DL, YX and Y-QK analyzed the data. S-TG, J-BZ, DL, K-HW and Y-QK wrote and revised the manuscript. All authors have read and approved the current version of the manuscript.

This work was partly supported by the National Natural Science Foundation of China (81660094), the Fund for Yunling Scholar (YLXL20170002), the General Joint Project of the Department of Science and Technology of Yunnan Province and Kunming Medical University (2017FE467 (-038), and 2017FE467(-130)), the Project for Innovation Team of Department of Science and Technology of Yunnan Province, China (2018HC005), the Fund of Department of Education of Yunnan Province (2019Y0352), the Fund of Health Commission of Yunnan Province (2018NS0085), the Fund of Yunnan Provincial Clinical Research Center for General Surgical Diseases (zx2019-03-03) and Yunnan Provincial Clinical Research Center for Skin Immune Diseases (2019ZF012) from Science and Technology Department of Yunnan Province, and the China Postdoctoral Science Foundation (2019M663580).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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