Saleh A. Naser

Dextran sodium sulfate (DSS) is widely used in vivo to induce epithelial injury; however, the epithelial-specific responses to DSS and mechanisms associated with barrier restoration remain incompletely understood. Here, we establish an in vitro DSS-induced epithelial damage model using differentiated Caco-2 cell monolayers. Exposure to 2.5%(w/v) DSS for 24 h resulted in significant upregulation of the pore-forming protein Claudin-2(CLDN2) (2.677-fold) and elevated oxidative marker protein levels of NADPH oxidase 1 (NOX1), accompanied by increased reactive oxygen species (ROS) production confirmed via dihydroethidium (DHE) staining. Increased epithelial permeability was observed by transepithelial electrical resistance (TEER) analysis, without significant cytotoxicity. Surprisingly, ELISA analysis revealed significant downregulation of Interleukin-6 (IL-6) (0.364 ± 0.034 pg/mL) following DSS exposure. Treatment with 1 ng/mL recombinant IL-6 (rIL-6) improved epithelial barrier function during continued DSS exposure, whereas a higher dose of 5 ng/mL did not produce the same restorative response. Furthermore, treatment of DSS-damaged Caco-2 monolayer with 10 mM butyrate significantly improved epithelial barrier function. Collectively, this study establishes a reproducible DSS-induced Caco-2 epithelial injury model and demonstrates concentration-dependent effects of rIL-6 and barrier-restorative effects of butyrate, providing an epithelial-specific platform for investigating intestinal barrier dysfunction relevant to IBD.

Crohn’s disease (CD) is a chronic inflammatory bowel disease with a rising incidence and prevalence worldwide. It is associated with Mycobacterium avium subspecies paratuberculosis (MAP). Current CD treatment strategies are based on anti-inflammatory therapies, including anti-TNF-α drugs. These treatment options provide short-term benefits and are associated with numerous side effects in CD patients. Manuka honey is distinguished from other honey by its high content of methylglyoxal (MGO). MGO, a reactive metabolite, is also generated endogenously in macrophages during infection through glycolysis; however, the amount is insufficient to neutralize the ongoing infection and subsequent tissue damage. This study examined whether exogenous, low-dose MGO can modulate MAP-driven inflammatory and glycolysis- and lactate-associated markers in infected macrophages. THP-1 macrophages were infected with the CD-associated MAP strain and then treated with MGO doses at defined time intervals. We measured markers of M1-/M2-like phenotype polarization, monocarboxylate transporters, lactate export, antioxidant responses, cytokines, and selected glycolysis- and lactate-associated markers at both the mRNA and protein levels. MGO reduced M1 signaling markers CXCL10 (p < 0.05), TNF-α (p < 0.0001), IL-1β (p < 0.01), and IL-6 (p < 0.0001). Simultaneously, MGO promoted M2 shift, elevating CD206 by 1.20-fold and IL-10 by 7-fold. Low-dose MGO administration was associated with increases in Nrf-2 (1.4-fold), HO-1 (1.4-fold), and IL-1Ra (1.5-fold), while the pro-inflammatory cytokines decreased. Metabolically, MGO downregulated MCT4 (p < 0.01) and reduced lactate export by 30%. These changes were coupled with higher PHD2 (1.4-fold) and decreases in GLUT1 (0.9-fold), PKD1 (0.8-fold), and IL-1β, consistent with attenuated glycolysis- and lactate-associated inflammatory signaling. These results suggest that hormetic concentration of MGO mitigates MAP-induced inflammatory activation while altering glycolysis- and lactate-related signaling markers in infected macrophages. Most importantly, we unraveled the predicted molecular mechanism by which MGO suppresses inflammation and modulates oxidative damage.

Background: Infection with Mycobacterium avium paratuberculosis (MAP) is closely associated with Crohn’s disease (CD) development, where excessive inflammation and marked intestinal damage are observed. Objectives: In this study, the role of short chain fatty acids, including propionic acid (PPA) and butyric acid (BA), was evaluated in an in vitro model, mimicking CD characteristics. Methods: MAP-infected THP-1 macrophages were treated with 1 mM and 10 mM of PPA or BA, and the conditioned media was co-cultured in Caco-2 cells. Results: Both PPA and BA caused an M2 shift with significant downregulation (p-value < 0.0001) in pro-inflammatory markers at both the RNA and protein levels. The downregulation is most likely due to the antimicrobial properties of PPA and BA. MAP growth was inhibited by several folds in MGIT (Mycobacteria Growth Indicator Tube) culture media supplemented with PPA or BA. Dysfunctional Caco-2 intestinal epithelial cells’ integrity and function, due to MAP infection, were restored with PPA and BA treatment. Specifically, NOX1 expression was significantly decreased in 10 mM of PPA or BA-treated cells (p < 0.001), as validated by RT-PCR and microscopy. PPA and BA restored tight junction integrity by decreasing Claudin-2 expression in the MAP group. Conclusions: The data clearly demonstrated that short chain fatty acids contain anti-inflammatory and antimicrobial properties with downstream beneficial effects on damaged intestinal epithelial cells, suggesting potential benefits as a dietary supplement for CD patients, particularly those who are not pregnant, due to a possible increased risk of autism spectrum disorder (ASD) development in offspring associated with propionic acid exposure.

We previously demonstrated that propionic acid (PPA), a common food preservative and a metabolic byproduct of bacteria in dysbiosis (as seen in individuals affected with autism spectrum disorder, ASD), can lead to increased brain astrocyte proliferation and neuroinflammation in mice. We also showed that in vitro treatment of human neural stem cells with PPA increased glial cell vs. neuron differentiation and yielded a marked increase in pro-inflammatory cytokines. In this study, a group of mice (FVB/N-Tg(GFAPGFP)14Mes/J) was exposed in utero through the maternal diet and postnatally ad libitum to a PPA-rich diet, while the control group was fed a normal diet. Intestinal tissue from offspring mice at 1 month (1 M) and 5 months (5 M) were then studied for neurodifferentiation and gastrointestinal (GI) inflammation. There was a significant increase in GFAP (Glial fibrillary acidic protein) expression (1.5427-fold and 1.6097-fold in 1 M and 5 M, respectively) and GFAP protein levels (1.5616-fold and 1.6701-fold in 1 M and 5 M, respectively) in the PPA group mice. It is worth mentioning that the expression level of intestinal astrocyte markers in the PPA group was significantly and multi-fold lower than that in the brain tissue. Contrary to data from brain tissue, the expression of pro-inflammatory cytokines in the PPA group decreased in intestinal tissue at 5 M (IL-6: 0.4403-fold; TNF-α: 0.4007-fold), while IL-10 expression and protein levels increased (1.9360-fold and 1.3428-fold, respectively). The data demonstrates that although there was a significant increase in GFAP in the intestine suggesting gliosis, there was an overall anti-inflammatory cytokine profile. The effect of PPA on intestinal cytokines is most likely in part due to the lower expression of GFAP in the enteric nervous system than the central nervous system (and the lower number of intestinal glia than astrocytes in the brain) and the dominance of intestinal macrophages and other immune cells compared to that in the brain. The overall finding strongly suggests that the PPA-rich diet affects the enteric glia state as shown by an increase in GFAP; however, it maintains the overall anti-inflammatory cytokine profile, possibly due to M2 macrophage polarization.

Corrigendum on: Alhendi, Ala, and Saleh A. Naser. "MCT4 inhibition attenuates inflammatory response to Mycobacterium avium paratuberculosis infection and restores intestinal epithelial integrity in vitro." Frontiers in Immunology 16: 1562100. Error in Figure/TableIn the published article, there was an error in Figure 4 as published. Wrong figure (old version of figure 5 before reviewer’s suggestion) is inserted in place of actual figure 4. The corrected Figure 4 and its caption appear below. Figure 4Inhibition of MCT4 in THP-1 cells during MAP infection restores basal intestinal cell oxidative status and mucin production. The supernatants of THP-1 cells under different conditions of CHCα treatment and MAP infection were collected 24 h after infection. These were used to substitute the culture media for growing and differentiated HT-29 cells for 24 h before RNA extraction. (A) qRT-PCR analysis of NOX-1 expression in HT-29 cells (n = 3). (B) qRT-PCR analysis of MUC2 expression in HT-29 cells (n = 5). *Indicates P-values less than 0.05, **indicates P-values less than 0.005, and ***indicates P-values less than 0.001The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.

Introduction: Mycobacterium avium paratuberculosis (MAP) plays a significant role in Crohn’s disease (CD). Monocarboxylate transporter 4 (MCT4) is a proton-coupled symporter of lactate that facilitates the inflammatory shift in macrophages and increases their reliance on glycolysis. MCT4 is also involved in the negative regulation of intestinal epithelial barrier function.Methods: In this in vitro study, we examined the role of MCT4 in macrophages and its effect on intestinal epithelial homeostasis during MAP infection. We used cultured THP-1 macrophages infected with a clinical strain of MAP (UCF4) as well as intestinal cell lines, Caco-2 and HT-29. MCT4 was inhibited using α-cyano-4-hydroxycinnamic acid (CHCα).Results: Infection of THP-1 cells with MAP upregulated MCT4 expression (2 folds) and resulted in a significant increase in lactate export (1.3 folds), TNFα (13.8 folds), and IL-6 (1.3) via TLR2 activation. Consequently, intestinal damage markers were also upregulated, including MUC2 (2.5 folds), NOX-1 (2 folds), SERPINE1 (2.1 folds), IL-6 (1.6 folds), and CLDN2 (1.4 folds). Inhibition of MCT4 during MAP infection with CHCα significantly reduced TNF-α and IL-6 levels. This effect on macrophages restored baseline oxidative status and mucin production in HT-29 intestinal cells. Moreover, MCT4 inhibition in a MAP-infected THP-1-Caco-2 co-culture system restored IL-6 and SERPINE1 to normal levels and enhanced tight junction protein, TJP1 (ZO-1), expression.Conclusion: Collectively, this study revealed the significant role of MCT4 in CD pathophysiology during MAP infection and highlighted MCT4 as a potential therapeutic target for CD treatment.

Like TNFα, IL-6 is upregulated in Crohn’s disease (CD) especially in patients associated with Mycobacterium avium paratuberculosis (MAP) infection, and both cytokines have been targeted as a therapeutic option for the treatment of the disease despite the accepted partial response in some patients. Limited response to anti-IL-6 receptor-neutralizing antibodies therapy may be related to the homeostatic dual role of IL-6. In this study, we investigated the effects and the signaling mechanism of IL-6 involved in intestinal epithelial integrity and function during MAP infection using an in vitro model that consists of THP-1, HT-29 and Caco-2 cell lines. Clinically, we determined that plasma samples from MAP-infected CD patients have higher IL-6 levels compared to controls (P-value < 0.001). In CD-like macrophages, MAP infection has significantly upregulated the secretion of IL-6 and the shedding of (IL-6R) from THP-1 macrophages, P-value < 0.05. Intestinal cell lines (Caco-2 and HT-29) were treated with the supernatant of MAP-infected THP-1 macrophages with or without a neutralizing anti-IL-6R antibody. Treating intestinal Caco-2 cells with supernatant of MAP-infected macrophages resulted in significant upregulation of intestinal damage markers including claudin-2 and SERPINE1/PAI-1. Interestingly, blocking IL-6 signaling exacerbated that damage and further increased the levels of the damage markers. In HT-29 cells, MAP infection upregulated MUC2 expression, a protective response that was reversed when IL-6R was neutralized. More importantly, blocking IL-6 signaling during MAP infection rescued damaged Caco-2 cells from MAP-induced apoptosis. The data clearly supports a protective role of IL-6 in intestinal epithelia integrity and function especially in CD patients associated with MAP infection. The findings may explain the ineffective response to anti-IL6 based therapy and strongly support a therapeutic option that restores the physiologic level of IL-6 in patient’s plasma. A new treatment strategy based on attenuation of IL-6 expression and secretion in inflammatory diseases should be considered.

This study, conducted between 4 October 2013, and 30 November 2018, tested the hypothesis that triple antimicrobial therapy, targeting Mycobacterium avium subspecies paratuberculosis (MAP), long considered a putative cause, would favorably affect Crohn’s disease. A double-blind multicenter study of adults with active Crohn’s disease, (i.e., Crohn’s Disease Activity Index [CDAI] 220–450 plus C-reactive protein ≥ 1.0 mg/dL, fecal calprotectin (FCP) >162.9 µg/g stool, or recent endoscopic or radiographic confirmation of active disease) receiving concomitant standard-of-care Crohn’s disease treatment (Clinicaltrials.gov: NCT01951326) were stratified by anti-tumor necrosis factor use and randomized (1:1) to anti-MAP RHB-104 (clarithromycin 95 mg, rifabutin 45 mg, and clofazimine 10 mg per capsule) (n = 166), resulting in clarithromycin 950 mg/day, rifabutin 450 mg/day, and clofazimine 100 mg/day, or placebo (n = 165) for up to 52 weeks. A greater proportion of RHB-104 versus placebo-treated patients met the primary endpoint—remission (i.e., CDAI < 150)—at week 26 (36.7% [61/166] vs. 22.4% [37/165], respectively; 95% CI for difference: 4.6, 24.0, p = 0.0048; chi-square test). Clinical response (reduction of CDAI by ≥100 points from baseline) at week 26 (first secondary endpoint) was also higher among the patients treated with RHB-104 (73/166 [44.0%]) compared with placebo (50/165 [30.3%]; 95% CI for difference: 3.4, 24.0, p = 0.0116), and it remained higher at week 52 among the patients treated with RHB-104 (59/166 [35.5%] vs. (35/165 [21.2%] for placebo; 95% CI for difference: 4.7, 23.9, p = 0.0042). A statistically significantly greater decline in FCP (another prospective efficacy endpoint) was also observed in RHB-104-treated patients, compared with placebo, at weeks 12, 26, and 52. The rates of serious adverse events were similar between groups (RHB-104: 18.7%; placebo: 18.8%). No patient died during the study. Antimicrobial therapy directed against MAP resulted in significantly greater improvement in clinical and laboratory (FCP) measures of active Crohn’s disease.

Evidence shows that Autism Spectrum Disorder (ASD) stems from an interplay of genetic and environmental factors, which may include propionic acid (PPA), a microbial byproduct and food preservative. We previously reported that in vitro treatment of neural stem cells with PPA leads to gliosis and neuroinflammation. In this study, mice were exposed ad libitum to a PPA-rich diet for four weeks before mating. The same diet was maintained through pregnancy and administered to the offspring after weaning. The brains of the offspring were studied at 1 and 5 months postpartum. Glial fibrillary acidic protein (astrocytic marker) was significantly increased (1.53 ± 0.56-fold at 1 M and 1.63 ± 0.49-fold at 5 M) in the PPA group brains. Tubulin IIIβ (neuronal marker) was significantly decreased in the 5 M group. IL-6 and TNF-α expression were increased in the brain of the PPA group (IL-6: 2.48 ± 1.25-fold at 5 M; TNF-α: 2.84 ± 1.16-fold at 1 M and 2.64 ± 1.42-fold, at 5 M), while IL-10 was decreased. GPR41 and p-Akt were increased, while PTEN (p-Akt inhibitor) was decreased in the PPA group. The data support the role of a PPA-rich diet in glia over-proliferation and neuro-inflammation mediated by the GPR41 receptor and PTEN/Akt pathway. These findings strongly support our earlier study on the role of PPA in ASD.

Background: Vitamin D plays a vital role in modulating both innate and adaptive immune systems. Therefore, vitamin D deficiency has been associated with higher levels of autoimmune response and increased susceptibility to infections. CYP27B1 encodes a member of the cytochrome P450 superfamily of enzymes. It is instrumental in the conversion of circulating vitamin D (calcifediol) to active vitamin D (calcitriol). This is a crucial step for macrophages to express Cathelicidin Anti-microbial Peptide (CAMP), an anti-bacterial factor released during the immune response. Our recent study indicated that a Crohn’s disease (CD)-associated pathogen known as Mycobacterium avium paratuberculosis (MAP) decreases vitamin D activation in macrophages, thereby impeding cathelicidin production and MAP infection clearance. The mechanism by which MAP infection exerts these effects on the vitamin D metabolic axis remains elusive. Methods: We used two cell culture models of THP-1 macrophages and Caco-2 monolayers to establish the effects of MAP infection on the vitamin D metabolic axis. We also tested the effects of Calcifediol, Calcitriol, and SB203580 treatments on the relative expression of the vitamin D metabolic genes, oxidative stress biomarkers, and inflammatory cytokines profile. Results: In this study, we found that MAP infection interferes with vitamin D activation inside THP-1 macrophages by reducing levels of CYP27B1 and vitamin D receptor (VDR) gene expression via interaction with the TLR2-dependent p38/MAPK pathway. MAP infection exerts its effects in a time-dependent manner, with the maximal inhibition observed at 24 h post-infection. We also demonstrated the necessity to have toll-like receptor 2 (TLR2) for MAP infection to influence CYP27B1 and CAMP expression, as TLR2 gene knockdown resulted in an average increase of 7.78 ± 0.88 and 13.90 ± 3.5 folds in their expression, respectively. MAP infection also clearly decreased the levels of p38 phosphorylation and showed dependency on the p38/MAPK pathway to influence the expression of CYP27B1, VDR, and CAMP which was evident by the average fold increase of 1.93 ± 0.28, 1.86 ± 0.27, and 6.34 ± 0.51 in their expression, respectively, following p38 antagonism. Finally, we showed that calcitriol treatment and p38/MAPK blockade reduce cellular oxidative stress and inflammatory markers in Caco-2 monolayers following macrophage-mediated MAP infection. Conclusions: This study characterized the primary mechanism by which MAP infection leads to diminished levels of active vitamin D and cathelicidin in CD patients, which may explain the exacerbated vitamin D deficiency state in these cases.